Presentation Information
[P01-101]Engineering Iron–Sulfur Cluster Supply to Improve Iron–Sulfur Protein Function for Metabolic Production in Escherichia coli
○Akito Odani1, Takumi Shirakawara1, Hidenobu Hirayama1,2, Ryota Hidese1,2, Akihito Kondo1,2,3, Toshihide Matsuno4, Jun Ishii1,2,3 (1. Graduate School of Science, Technology an Innovation, Kobe University (Japan), 2. Engineering Biology Research Center Kobe University (Japan), 3. Graduate School of Engineering Faculty of Engineering, Kobe University (Japan), 4. National Institute of Technology, Fukui College (Japan))
Keywords:
Iron-sulfur protein,E.coli,MEP pathway,iscR gene,oxyR gene,lycopene
[Purpose]
This study aimed to develop an Escherichia coli strain capable of high-level production of iron–sulfur (Fe–S) proteins, which are essential for the biosynthesis of isoprenoids such as lycopene. Because Fe–S proteins are highly sensitive to oxygen, their intracellular abundance is limited, making them a rate-limiting factor in lycopene biosynthesis.
[Methods]
Since the expression and activity of the Fe–S proteins IspG and IspH in the MEP pathway are difficult to measure directly, lycopene biosynthesis genes (crtE, crtB, and crtI) were introduced into E. coli to convert isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) into lycopene. Lycopene production was used as an indirect indicator of Fe–S protein activity and quantified spectrophotometrically.
To enhance Fe–S cluster availability, an iscR knockout strain was constructed to relieve transcriptional repression of Fe–S cluster biosynthesis genes. In addition, an oxyR overexpression strain was developed to promote antioxidant enzyme expression and thereby suppress oxidative degradation of Fe–S clusters.
[Results]
Lycopene production in the iscR knockout strain increased by approximately 1.45-fold compared to the wild-type strain (BW25113). Similarly, the oxyR overexpression strain showed a 1.41-fold increase in lycopene production. These results indicate that both genetic modifications enhanced Fe–S protein-dependent metabolic flux.
[Consideration]
The iscR disruption likely alleviated transcriptional repression of Fe–S cluster biosynthesis genes, thereby increasing Fe–S cluster supply. Meanwhile, oxyR overexpression likely enhanced antioxidant defenses, reducing Fe–S cluster degradation. Together, these strategies improved Fe–S protein availability, relieving rate-limiting steps in the pathway.
[Conclusion]
Enhancing Fe–S cluster biosynthesis and preventing their degradation are effective strategies for improving lycopene production in E. coli. These findings provide a general approach for enhancing the biosynthesis of Fe–S protein-dependent compounds and may be broadly applicable to microbial production of valuable metabolites.
This study aimed to develop an Escherichia coli strain capable of high-level production of iron–sulfur (Fe–S) proteins, which are essential for the biosynthesis of isoprenoids such as lycopene. Because Fe–S proteins are highly sensitive to oxygen, their intracellular abundance is limited, making them a rate-limiting factor in lycopene biosynthesis.
[Methods]
Since the expression and activity of the Fe–S proteins IspG and IspH in the MEP pathway are difficult to measure directly, lycopene biosynthesis genes (crtE, crtB, and crtI) were introduced into E. coli to convert isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) into lycopene. Lycopene production was used as an indirect indicator of Fe–S protein activity and quantified spectrophotometrically.
To enhance Fe–S cluster availability, an iscR knockout strain was constructed to relieve transcriptional repression of Fe–S cluster biosynthesis genes. In addition, an oxyR overexpression strain was developed to promote antioxidant enzyme expression and thereby suppress oxidative degradation of Fe–S clusters.
[Results]
Lycopene production in the iscR knockout strain increased by approximately 1.45-fold compared to the wild-type strain (BW25113). Similarly, the oxyR overexpression strain showed a 1.41-fold increase in lycopene production. These results indicate that both genetic modifications enhanced Fe–S protein-dependent metabolic flux.
[Consideration]
The iscR disruption likely alleviated transcriptional repression of Fe–S cluster biosynthesis genes, thereby increasing Fe–S cluster supply. Meanwhile, oxyR overexpression likely enhanced antioxidant defenses, reducing Fe–S cluster degradation. Together, these strategies improved Fe–S protein availability, relieving rate-limiting steps in the pathway.
[Conclusion]
Enhancing Fe–S cluster biosynthesis and preventing their degradation are effective strategies for improving lycopene production in E. coli. These findings provide a general approach for enhancing the biosynthesis of Fe–S protein-dependent compounds and may be broadly applicable to microbial production of valuable metabolites.
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